Jääskeläinen Tiina, Ryhänen Sanna, Mäenpää Pekka H
Department of Biochemistry, University of Kuopio, Kuopio, Finland.
J Cell Biochem. 2003 Aug 15;89(6):1164-76. doi: 10.1002/jcb.10572.
Abstract vitamin D receptor (VDR) and retinoid X receptor (RXR) heterodimerize to mediate the genomic actions of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3), calcitriol), the biologically active form of vitamin D(3). In this study, we show that 9-cis retinoic acid (9-cisRA), the ligand for RXR, accelerates calcitriol-induced expression of osteocalcin gene, the marker for mature osteoblasts. Calcitriol and its synthetic analog KH1060 (1 nM) induced osteocalcin secretion after a 96-h incubation period as detected by radioimmunoassay. When these compounds were used together with 9-cisRA, osteocalcin protein secretion was, however, detected already after 72 and 48 h, respectively. Detection of osteocalcin mRNA with quantitative PCR revealed elevated mRNA levels already after a 4-h treatment of the cells with calcitriol, KH1060, or 9-cisRA compared with untreated cells. In combination treatments, 9-cisRA rapidly stimulated osteocalcin mRNA synthesis induced by the different vitamin D(3) compounds. In MG-63 cells treated with calcitriol or KH1060, the stimulation was maximal after the first 4 h and diminished thereafter. In fact, after the 48-h incubation 9-cisRA reduced osteocalcin mRNA levels in KH1060-treated cells, the amount of mRNA being only 44% of the levels obtained with KH1060 alone. The reduction was accompanied by an increased degradation rate of both VDR and RXRbeta in the presence of 9-cisRA. Furthermore, 9-cisRA increased the formation of RXRbeta-VDR-VDRE complex on the osteocalcin gene VDRE. These results suggest that 9-cisRA accelerates calcitriol-induced osteocalcin production in human osteoblastic cells through increased formation of transcriptionally active chromatin complexes and, subsequently, promotes degradation of the heterodimeric complex of VDR and RXR.
摘要 维生素D受体（VDR）与视黄酸X受体（RXR）形成异源二聚体，介导1α,25 - 二羟基维生素D3（1α,25(OH)2D3，骨化三醇）的基因组作用，骨化三醇是维生素D3的生物活性形式。在本研究中，我们发现RXR的配体9 - 顺式视黄酸（9 - cisRA）可加速骨化三醇诱导的骨钙素基因表达，骨钙素是成熟成骨细胞的标志物。通过放射免疫分析检测，骨化三醇及其合成类似物KH1060（1 nM）在孵育96小时后诱导骨钙素分泌。然而，当这些化合物与9 - cisRA一起使用时，分别在72小时和48小时后就检测到了骨钙素蛋白分泌。用定量PCR检测骨钙素mRNA发现，与未处理的细胞相比，用骨化三醇、KH1060或9 - cisRA处理细胞4小时后，mRNA水平就已升高。在联合处理中，9 - cisRA迅速刺激了不同维生素D3化合物诱导的骨钙素mRNA合成。在用骨化三醇或KH1060处理的MG - 63细胞中，刺激作用在最初4小时后达到最大，此后逐渐减弱。事实上，在孵育48小时后，9 - cisRA降低了KH1060处理细胞中的骨钙素mRNA水平，mRNA量仅为单独使用KH1060时的44%。这种降低伴随着在9 - cisRA存在下VDR和RXRβ降解速率的增加。此外，9 - cisRA增加了骨钙素基因维生素D反应元件（VDRE）上RXRβ - VDR - VDRE复合物的形成。这些结果表明，9 - cisRA通过增加转录活性染色质复合物的形成来加速骨化三醇诱导的人成骨细胞中骨钙素的产生，随后促进VDR和RXR异源二聚体复合物的降解。
